Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Raymond Delashmitt
Abstract
The goal of this project was to characterize the WetLabs FLNTUSB fluorometer and
determine the possibility of using it as an instrument to measure wild algal growth on substrates
in vivo. To do this several aspects were investigated, which include determining the angle
sensitivity of the instruments, if the instruments were able to compared directly to each other, if
the signals recorded demonstrated that the instruments were recording actual fluorescence of
algae, and to correlate the signal recorded to harvest data during the same time period. The
results of this investigation showed that the angle sensitivity depends on whether the angle from
normal is within the beam plane created by the LED and absorption cones, or if it was
perpendicular to the beam plane. In the investigation to determine if the fluorometers were
observing actual fluorescence of chlorophyll in the algae, it was determined that the signal was
from fluorescence due to a photo-inhibition effect and the variance being dependent on the size
of the signal. Finally, there is evidence that the fluorescence observed during the deployments of
this project can be compared to harvest data during the same time period and the relative changes
in both of these data sets appear to match, especially during periods that the substrates the
fluorometers were observing were cleaned and the signal dropped accordingly.
Fluorometer Background
Fluorometers have been widely used since the early 1970’s in marine biology research
applications and it has been proven they can accurately model the level of chlorophyll in the
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
water. It has also been shown previously that by detecting the chlorophyll, the fluorometer can
measure a sample’s level of ambient algae in water.1 This technique of measuring algae growth
has become a popular and widely accepted process due to the fact that the measurement can be
done accurately, in real-time, without needing to remove the algae from the environment, and
with a handheld instrument. Previous solutions to measuring algae involved removing the algae
from the water by taking water samples, then measuring the algae populations in a lab with
counting chamber methods2 or High Performance Liquid Chromatography3.
The fluorometer measures the level of chlorophyll by the amount that the object in the
beam fluoresces. The meter sends out a LED light of wavelength 470 nm, and when it comes in
contact with the chlorophyll a in the algae it is absorbed, exciting it to a higher quantum state
that then emits a photon back out at a wavelength of 695 nm4. This process is shown in figure 15
below, with the LED as the transmitter, and the algae represented as the chlorophyll a molecules.
Figure 1
Diagram showing absorption of the LED photon and reemission of a photon to the detector
1
(Aberle, September 2006)
(H Utermoehl, 1958)
3
(Schroeder, 1994)
4
(Wetlabs, 23 Dec 2009)
5
(SCCF Recon, 2010)
2
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
The data from the fluorometer is recorded as a digital count or an analog voltage. The
digital counts range from 50 to 4130, and the analog ranges from .072 V to 4.98 V. This means
that the fluorometers feature a dark count of 50, which is present in all data collected, and in all
the graphs featured, will have this value subtracted to give only the signals received by the
fluorometers.
Additionally, due to the nature of the fluorescence of the chlorophyll, an issue with the
detection is that the photon emitted from the algae is emitted in a random direction. Thus, the
meter is set up to do an average of a set number of samples to decrease the noise in the signal. In
these samples a variance is expected, which should be related to the signal strength by a square
root function. For all of the samples taken during the experiments, the average number of data
values taken before generating a single point was 60, then in the analysis of the data for hourly
and daily averages each of these single points were used.
Lab Experiments
For the controlled lab tests to measure the configuration of the fluorometers, the
experiments were to measure the angle of the local maxima of the signal produced by the
instruments when exposed to a point-like source of fluorescence and to measure the angles of
dispersion of the LED source and the cone of detection. To measure the angles of dispersion of
the LED, a reflective surface was used to allow tracing of the light cone, which resulted in the
following measurements in figure 2.
This recorded line was of the sharp edge the beam
dispersed by the LED, with the intensity dropping off substantially outside of the 15 degrees
recorded. This area of light shall be referred to as the maximum LED cone.
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 2
Diagram of the LED and absorption cones
The colored portions of the diagram show the areas that are the maximum LED dispersion or
detector absorption cones. The maximum light dispersion cone was measured, while the cone of
maximum absorption was based off of orientation of the detector with the assumption of it
behaving in reverse to the LED photon dispersion beam. The absorption cone was drawn based
on the symmetry to the light cone and geometry of the detector offset from normal. These cones
are not the only areas that signal is detected by the fluorometer due to the Gaussian decay of the
signal. This decay of the signal allows the tails of the two cones to intersect in front of the face
of the instrument, which accounts for signal recorded by the fluorometer in the angle study
experiment. In the diagram, it shows how when the angle is not in the plane where the beams
cross, the theoretical dispersion is the same, and should result in the least amount of distortion of
the signal. When the angle is measured in the beam plane, the geometry shows the cones of the
LED and detector with an offset of 15 degrees towards each other, and each of the cones having
a 15 degrees spread. This dispersion results in a theoretical range between .5 cm and 1.8 cm
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
where the signal is maximum, which is shown in later experiments to result in an oversaturated
signal.
The next experiments were to determine the sensitivity of the fluorometers to a change in
angle when the distance was held constant. The first experiment was to use a point-like source
of 1 cm radius as the source of the fluorescence and to go through the entire range of angles
possible to the fluorometers. The experiment had the change of angle both orthogonal to and
within the plane that the LED and detector cone beams intersect. In this setup, the data was
taken at intervals of 10 degrees while the radial distance of the face of the fluorometer to the
substrate was held constant. The range of angles represented express the range of freedom that
the biowiper and the size of the fluorometers allow, generally 70 degrees from normal in either
direction.
The data for when the angle was perpendicular to the plane resulted with the absolute
maximum at 90 degrees to the substrate with the signal decaying as the angle deviated from this
value. On the closer distances the physical offset of the LED and detector, in the design of the
instrument to account for the biowiper, affected my ability to keep the same distance on either
side from normal. This resulted in the curve being biased towards the angles where the open
biowiper is farthest away from the substrate due to its relative closer proximity to the substrate.
The data from this experiment is featured in figure 3 below with the dark counts accounted for in
the signal.
The next aspect was when the angle was in the beam plane where the data showed signs
from the previous analysis of the local maxima of the signal, but with the maximum directly in
front of the LED beam being much larger than the other maxima present in the signal. The
secondary maximum that was observed in some of the signals was towards the theoretical beam
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
that is directly in front of the detector, and is greatest when the distances to the substrate are
smallest. The maximum of the signal shifted as the distances away from the substrate changed.
This shows evidence of an interaction between the LED and detectors cones that pulling of the
maxima towards normal is occurring, due to the decay tails limiting the amount of photons
available to be detected along the previously stated angles. At the smallest distance of 3 cm
away, the maximum was at 20 degrees from normal towards the LED beam side. At the larger
distances of 5, 7, and 8 cm away, the maximum was between 40 and 50 degrees from normal,
towards the LED beam side. The other signals are between the 20 – 50 degrees from normal
towards the LED beam side. This data of this experiment is featured in below in figure 4 and
features the complete dataset with 6 points taken for each angle to show the spread of data for
certain angles and a subtraction of the dark counts.
Figure 3
Angle study with angle perpendicular to beam plane and viewing point source
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 4
Angle study with angle within beam plane and viewing point source
The second aspect of the angle analysis that was done was to repeat the previous test of
the signals vs. angles, but have the fluorometers observing a substrate that would appear to be an
infinite plane of fluorescence. The substrate used was previously shown to be similar to algae
fluorescence at similar distances when tested with the fluorometers normal to the substrate. The
signal curves of the fluorometers were higher than the point tests, and were smoother and more
level than the point tests. This leveling can be explained by the averaging effect of allowing the
instrument to view fluorescent points closer and further away than the point source experiment.
For the test with the angle perpendicular to the beam plane, the signal showed a
decreased decay of the signal at large angles from normal to the substrate, and in comparison the
difference was between 10 and 40 percent of the average for the distance. The curves shown in
figure 5 that appeared were more level than the point source, and mostly just showed the
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
decreasing trend from the physical offset on the instrument. This seems to suggest that when the
fluorometer is set up with the angle perpendicular to the beam plane, the signal is fairly constant
with respect to the angle from the substrate, so long as the angle is at or within 45 degrees from
normal.
Additionally, when the angle is taken within the beam plane, the curve once again is
smoother, but retains the maxima seen in the point test. The absolute maximum of the graph
seems to be offset more towards 45 degrees from normal, towards the LED beam side.
Throughout the distances, the absolute maximum ranges from 30 degrees to 50 degrees for
distances of 4 cm and 8 cm respectively. This suggests that if the fluorometer is set up with this
orientation the ideal angle will be around 45 degrees when the substrate is between 4 to 8 cm
directly out from the face of the instrument. This set of data is shown below in figure 6 with an
account for the dark counts in the signal.
Figure 5
Angle study with angle perpendicular to beam plane and viewing infinite plane
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 6
Angle study with angle within beam plane and viewing infinite plane
The results seem to show that for the constrictions of setup on the York River Flume,
having the 45 degrees from normal will be possible when the angle is both in the beam plane and
perpendicular to it. The difference is that when the angle is in the beam plane, and the angle is
towards the LED beam side, the fluorometer will be detecting chlorophyll readings from a more
concentrated area of the substrate. When the angle is perpendicular to the beam plane, then it
should result in an averaging effect of the substrate, with the area of view being larger than the
other setup. Additionally, the data suggests that with the angle perpendicular to the beam plane,
if another angle is desired it should be able to perform at a nearly equal level, while when the
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
angle is within the beam plane the peak performance is limited to between 30 and 50 degrees
from normal towards the LED side.
York River Experiments
The first experiment that was done on the York River platform concerning the
fluorometer data was to test whether the fluorometer signals would be comparable when they
were observing the same substrate. To test this, the fluorometers were deployed from November
4-8 as shown in figure 7, with them at 45 degrees to the substrate with the angles perpendicular
to the beam plane, so that the signal readings would be affected least by the angle of deployment.
Additionally, due to space constrictions on the platform, the fluorometers were deployed
observing opposite sides of the same substrate since there was no indication of an algae growth
difference between the different sides.
Figure 7
Diagram showing physical setup of fluorometers for comparison and extended deployments
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
The following data was recorded by both of the fluorometers, and are represented by the
averages of each hour with error bars of a single standard deviation. The fluorometers feature a
dark count of 50 counts, and in the follows graphs that values is subtracted away to give only the
values that are recorded by the fluorometers. This data is too short of a deployment to determine
cyclic behavior of the algae, but it does show that despite best efforts being made to deploy the
fluorometers in the same orientation and distance from the substrate, the sensitivity of the
instruments is too great. The difference between the curves can most likely be attributed to the
sensitivity to distance that follows a decaying exponential curve.
This shows that the
fluorometers can’t be mounted so that they give absolute readings on the signal detected from the
algae, especially to the level of comparing between two different fluorometers. Although the
fluorometers are still useful in this setup to determine the relative change of the signal detected
over a deployment. The following graphs show the signal recorded during this deployment, and
show the daily average in figure 8, the variance per hour in figure 9, and factional variance in
figure 10, with each of these graphs using data without the dark counts of the instruments.
Figure 8
Comparison of recorded hourly averages of fluorometer data from November 4 - 8
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
In the following figure 9, the data of the variance over an hour for each fluorometer is
shown. The large variance shown during the first day for the right fluorometer corresponds to an
unusually large signal in the comparison, suggesting that the data during that time period was in
the presence of an unrecorded variable. The large variance when compared to the size of the
signal generated also suggests that the variance seen in this sample is due to the photon
collection rather than instrument error, because it varies with the size of the signal. This suggests
that the cause of the large initial spike was from a variable such as algae from farther down the
flume breaking off and impeding the view of the fluorometer.
The floating algae would
fluoresce the same as the algae on the substrate and it was floating in the water it would be closer
to the instrument and cause this large appearance of growth. Additionally, since the large
anomaly is present only for a few hours it suggests that the algae passed through the flume.
Figure 9
Comparison of recorded hourly average variance of fluorometer data from November 8 - 22
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
In figure 10, the fractional variance of the signal is represented, with it being the standard
deviation divided by the average signal for that hour. This shows how the variance is related to
the signal with the variance mostly within 5 percent of the signal. This suggests that the using
the average of the signals collected each hour is valid, and this is verified by similar results in
later deployments.
Figure 10
Comparison of recorded hourly average fractional variance of fluorometer data from November 8 - 22
The next experiment was an initial extended deployment of the fluorometer, which was
done on November 8 – 22. The deployment retained the setup from the previous comparison
experiment, but with only the left fluorometer used due to complications with the right
fluorometer. During this deployment, the structure of the signal showed definite signs of cyclical
behavior. This structure is shown in the following figure 11 with the dark counts removed and
follows a 24 cycle, with both the maximum and minimum represented between 11 AM and 2
PM. A strong explanation to this can come from photo-inhibition of the chlorophyll. PhotoPage 13 of 36
Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
inhibition is occurs when chlorophyll is oversaturated by a light source, in this case the sun, and
any attempts to further excite the chlorophyll results in a lower than expected reading. This
explains why there in a minimum in the signal when compared to readings taken at midnight,
when there is no sun. Another sign to confirm this photo-inhibition is a decrease in the variance
of the signal, which is discussed in the next section.
Figure 11
Recorded hourly averages of fluorometer data from November 8 - 22
The following figures show the variance and fractional variance of the data with the dark
counts removed. The variance in figure 12 shows the hourly variance of the data for this
deployment, and shows similar structure to the comparison experiment. Initially the variance
appears differ greatly throughout each day, but that is due to the large signal differences due to
the cyclic nature of the chlorophyll. This is confirmed through figure 13, which takes into
account the relationship of the variance to the signal it is from, and shows that most of the data
has a variance of less than 5 percent of the signal. Additionally, the variance confirms the
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
previous theory that photo-inhibition occurs due to the low variance during periods of highest
sun activity, around noon. This is shown again in figure 14 with the average variance per hour.
Figure 12
Recorded hourly average variance of fluorometer data from November 8 - 22
Figure 13
Recorded hourly average fractional variance of fluorometer data from November 8 - 22
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
In the following graphs the same data is used as above, with the variances averaged for
each hour. By looking at the average variance per hour in figure 14 the trend of lower variance
at high solar activity times is more apparent, shows a drop in the variance, while during the rest
of the day the average variance is fairly constant. The drop in the variance during this period
gives further evidence of photo-inhibition over saturating the chlorophyll, because as the
variance decreases it shows that the chlorophyll’s ability to fluoresce is inhibited by giving a
more constant reading. The fractional variance shown in figure 15 shows flattening similar to
the fluorometer comparison, but the noon drop is still present despite the removal of the factor of
variance related to signal strength.
Figure 14
Recorded variance per hour of fluorometer data from November 8 - 22
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 15
Recorded fractional variance per hour of fluorometer data from November 8 - 22
Another aspect that must be investigated of this deployment is if there is a single value in
the original signal data that correlates with the average of the day, which would allow a single
value to represent the dataset against the harvest yields. The data shows both a large maximum
and minimum between 11AM and 2 PM that is most likely caused by photo-inhibition, and this
gives reason to not use these extremes due to the large jump between them.
In order to
compensate for this, taking the recordings at night would give the most consistent signal
readings. In figure 16 the averages for each day are shown in comparison to the hourly averages
and the values that are closest to the averages are the values taken at 2 AM. To show this the
daily average is compared to just the 2 AM hour averages in figure 17. This relationship
between the 2 AM and the daily average is also present in the next deployment done in
December. This combined with the variance data showing that photo-inhibition occurs mostly at
noon, and thus to remove this factor a time would be needed that was opposite this time, which 2
AM fits into.
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 16
Comparison of recorded hourly vs daily averages of fluorometer data from November 8 - 22
Figure 17
Comparison of recorded 2 AM vs daily averages of fluorometer data from November 8 - 22
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
This final aspect of the deployment is shown in figure 18 and features the 2 AM hourly
data with dark counts removed and how it relates to harvested data taken from flume. The data
on the harvest was taken from an average of all of the screens that were related to the screen that
the fluorometer was directed towards. The first two points would be expected to match with the
fluorometer signal due to both of them starting with a fresh experiment and harvest. The third
point of harvest though is one of continuous growth throughout the entire period, while the
screen that the fluorometer was harvested and cleaned when the second point was taken. This
data is still useful because the relative growth rates between the days of 18 to 22 and the relative
change in the signal during that same period are similar, with the rates being slower than the rest
of the deployment. Additionally, after the second harvest when the screen the fluorometer was
observing was harvested clean, the fluorometer signal drops accordingly. This drop in the
fluorometer signal is a significant display that the fluorometer is in fact observing the
fluorescence of the algae rather than the fluorescence of the substrate or the reflection of the
original LED beam.
Figure 18
Comparison of recorded 2 AM of fluorometer data against harvest data from November 8 - 22
Page 19 of 36
Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
The next focus of the experiment was to run another extended trial of the fluorometers in
the same environment of the York River platform. The deployment was from December 1 – 13
and featured the same setup as the first, with the fluorometer set at 45 degrees to the substrate
with the angle perpendicular to the beam plane.
In this deployment the following data was
recorded, which shows similar structure to the first deployment with noon having the extremes of
the hourly data. In the following figure 19 the hourly averages are again shown with error bars
of the standard deviation of the hour and the dark counts removed.
Figure 19
Recorded hourly averages of fluorometer data from December 1 - 13
In the next two graphs the hourly variance and fractional variance is featured from this
data without the dark counts. In figure 20 it shows that the hourly variance decreases during the
times of largest solar activity, which suggests again that photo-inhibition is occurring. Due to
unknown causes the averages in this deployment have more outliers, though the variance largely
does not show anomalies. The lack of anomalies suggests that the outliers are due to the
substrate or setup moving, and not due to an instrument error, because the variance is similar to
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
the other samples and is only increased by the relationship between variance and signal strength.
These aspects are also present in the fractional variance along with how a large proportion of the
variances are within 4 percent of the signal average.
Figure 20
Recorded hourly average variance of fluorometer data for December 1 - 13
Figure 21
Recorded hourly average fractional variance of fluorometer data from December 1 - 13
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Another display of the data confirming the photo-inhibition comes from the following
figures of the variance and fractional variance per hour in figure 22 and figure 23. The both of
these datasets show at noon the variance decreases where the other hours have fairly constant
values. The variance is not as clean as the November deployment, but that can possibly be
explained due the flume being towed into the boatyard during the first days of the deployment,
and the additional disturbances that the flume would experience due to the closer proximity to
the shore and other working vessels. An interesting aspect is how despite this, the signals
recorded and the variances of the data don’t differ greatly from the previous undisturbed
deployment. This suggests that so long as the substrate and fluorometer are in a fixed position to
each other, the other factors occurring on the flume may be less important than previously
believed.
Figure 22
Recorded variance per hour of fluorometer data from December 1 - 13
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 23
Recorded fractional variance per hour of fluorometer data from December 1 - 13
Next, there was to find a time of the day that would be able to represent in the future,
what the average of the day would be closest to. When the daily average was placed on the same
plot as all of the hourly averages in figure 24 and figure 25 with the dark counts removed, the 2
AM value as previously found was again a best fit. Again both the maximum and minimum are
present in the data within an hour of each other and thus it seems best to use the time of 2 AM to
represent the most consistent value to represent the day. The 2 AM time period doesn’t fit the
daily average quite as well as in the previous deployment, but the uncertainties of both of the
values remain within each other. The time that the two averages differ the most is directly after
the full harvest on the 6th, but the difference is resolved after that.
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 24
Comparison of recorded hourly vs daily averages of fluorometer data from December 1 - 13
Figure 25
Comparison of recorded 2 AM vs daily averages of fluorometer data from December 1 - 13
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
The final aspect of this deployment was to again compare the 2 AM hourly average found
to be able to represent a cleaner form of the deployment data to the harvest data taken of each
substrate that represented the screen the fluorometer was directed towards. In figure 26 the
comparison between the 2 AM without dark counts and harvest data is shown. Unfortunately the
harvest data available during this time period was of growth that was undisturbed growth for
longer than the substrate that the fluorometer was directed towards was able to grow. The
relative growth is still comparable, but not as relevant as during the November deployment for
direct comparison of growth rates. Also this deployment featured a point of December 6th when
the screen the fluorometer was directed towards was scraped clean to take the substrate back to
an initial state. This shows how the signal decreases immediately during that time similar to the
previous deployment, confirming how the fluorometer was observing fluorescence of algae.
Another interesting aspect of this is if the assumption that the fluorometer was observing the
algal growth, then the growth increased rapidly after the clean harvest, suggesting that the
harvest stimulated more growth of algae that fluoresced than there was previously on the
substrate.
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Figure 26
Comparison of recorded 2 AM of fluorometer data against harvest data from December 1 - 13
Conclusions
During this project I was posed with the objective to characterize the WetLabs
FLNTUSB fluorometer and to determine if it was possible to measure the fluorescence to plot
the growth of wild algae. I determined that there are two setups possible when deploying the
fluorometer, either having the angle of deployment in or perpendicular to the beam plane created
by the LED and Detector absorption cones. If the angle is within the beam plane then the largest
maximum and most stable angle will be around 45 degrees towards the LED beam side. If the
angle is perpendicular to the beam plane the angle sensitivity is decreased from the previous
setup, and the angle is able to handle whatever angle the physical constraints are, though an
angle of more than 45 degrees is not recommended.
Page 26 of 36
Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
The next aspect was to deploy the fluorometers for extended periods, and through doing
so I determined that the fluorometers are useful to measure relative changes in signal, that can be
correlated to fluorescence of the algae. They are not able to be compared directly to each other
due to the high sensitivity to the instruments based on distance and angle, though once installed
in a fixed position the sensitivity of the fluorometer seems to vary greatly when other variables
are changed in the experiment. The fluorescence of the chlorophyll is dependent on the photoinhibition effect, though if the daily measurements are taken around 2 AM the values recorded
are very close to representing the daily average without experiencing the period photo-inhibition.
Finally, there is evidence that the fluorescence observed by the fluorometers can be correlated to
the algae density of the substrate that it is directed towards.
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Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Data Tables, Lab Experiment - Angle Sensitivity
In Beam Plane With Point Source
5 cm
3 cm
4 cm
Signal
Angle (Counts)
130
2128
130
2099
130
1915
130
2035
130
1970
130
1935
130
1975
130
1963
130
1965
120
1982
120
2002
120
2113
120
2244
120
2192
120
2114
120
1945
110
2427
110
2470
110
2455
110
2451
110
2457
110
2456
110
2410
100
2353
100
2348
100
2355
100
2367
100
2366
100
2320
100
2177
90
2149
90
1999
90
1928
90
1910
90
1879
Signal
Angle (Counts)
140
934
140
962
140
965
140
959
140
954
140
965
140
965
130
1206
130
1222
130
1221
130
1235
130
1243
120
1176
120
1177
120
1250
120
1305
120
1317
110
1221
110
1229
110
1237
110
1232
110
1238
100
1041
100
1052
100
1054
100
1052
100
1067
90
967
90
962
90
1016
90
1019
90
1016
80
865
80
895
80
899
Signal
Angle (Counts)
140
838
140
831
140
825
140
835
140
839
140
851
130
912
130
930
130
943
130
950
130
945
130
951
120
861
120
828
120
848
120
860
120
868
120
876
110
718
110
657
110
655
110
687
110
692
110
696
100
643
100
589
100
587
100
584
100
586
100
583
90
547
90
506
90
490
90
488
90
489
6 cm
7 cm
Signal
Angle (Counts)
140
222
140
205
140
209
140
213
140
232
140
273
130
284
130
289
130
319
130
332
130
331
130
329
120
402
120
412
120
413
120
347
120
372
120
362
110
288
110
320
110
340
110
333
110
332
110
300
100
275
100
287
100
298
100
299
100
298
100
284
90
274
90
269
90
266
90
262
90
262
Signal
Angle (Counts)
140
310
140
314
140
318
140
320
140
321
140
315
130
259
130
234
130
251
130
275
130
277
130
278
120
250
120
245
120
246
120
243
120
252
120
251
110
236
110
222
110
220
110
220
110
220
110
219
100
205
100
189
100
185
100
187
100
186
100
185
90
160
90
139
90
128
90
124
90
123
Page 28 of 36
Raymond Delashmitt
2010 - 2011
90
90
90
80
80
80
80
80
80
70
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
50
1867
1857
1829
1602
1663
1832
1833
1829
1821
1594
1511
1705
1706
1697
1713
1530
1347
1484
1495
1483
1447
1255
530
796
899
832
845
870
931
Application of Fluorometers to Measure Wild Algal Growth In Vivo
80
80
70
70
70
70
70
60
60
60
60
60
50
50
50
50
50
40
40
40
40
40
40
898
902
815
815
815
813
812
688
681
681
679
676
507
490
488
477
468
325
332
341
340
337
213
90
80
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
490
400
370
396
397
396
397
352
311
312
315
314
313
265
235
234
231
227
225
187
164
165
166
165
166
142
115
124
129
129
130
90
80
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
230
205
208
207
208
209
202
178
177
175
174
173
162
145
144
143
141
140
124
108
108
106
105
103
87
70
75
76
75
75
72
90
80
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
127
123
112
112
113
115
120
110
100
107
107
107
107
92
88
89
90
89
89
83
77
77
77
76
75
65
69
69
69
68
69
Page 29 of 36
Raymond Delashmitt
2010 - 2011
3 cm
Signal
Angle (Counts)
120
2225
120
2231
120
2242
120
2245
120
2252
120
2265
110
2320
110
2334
110
2337
110
2339
110
2352
110
2364
100
2392
100
2394
100
2390
100
2387
100
2389
100
2399
90
2406
90
2407
90
2407
90
2407
90
2408
90
2388
80
2321
80
2317
80
2321
80
2319
80
2320
80
2300
70
2187
70
2206
70
2214
70
2225
70
2224
70
2195
60
2065
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Perpendicular to Beam Plane With Point Source
4 cm
5 cm
6 cm
Signal
Angle (Counts)
130
913
130
933
130
935
130
952
130
963
130
1060
120
1119
120
1120
120
1125
120
1143
120
1152
120
1168
110
1178
110
1182
110
1174
110
1174
110
1177
110
1166
100
1145
100
1144
100
1138
100
1135
100
1136
100
1127
90
1081
90
1052
90
1102
90
1173
90
1173
90
1162
80
1010
80
1007
80
1037
80
1070
80
1075
80
1076
70
1033
Signal
Angle (Counts)
130
486
130
489
130
488
130
477
130
476
130
483
120
487
120
485
120
485
120
483
120
484
120
481
110
482
110
479
110
476
110
477
110
470
110
473
100
475
100
469
100
467
100
467
100
467
100
467
90
443
90
440
90
441
90
441
90
442
90
441
80
393
80
395
80
395
80
397
80
398
80
396
70
348
Signal
Angle (Counts)
140
182
140
180
140
183
140
185
140
187
140
218
130
264
130
256
130
246
130
249
130
250
130
254
120
281
120
286
120
288
120
288
120
288
120
290
110
295
110
295
110
294
110
292
110
285
110
282
100
233
100
221
100
245
100
243
100
245
100
245
90
253
90
256
90
261
90
261
90
260
90
258
80
239
7 cm
Signal
Angle (Counts)
140
112
140
111
140
111
140
113
140
126
140
129
130
146
130
145
130
145
130
145
130
145
130
146
120
157
120
158
120
157
120
157
120
158
120
159
110
161
110
162
110
162
110
160
110
160
110
160
100
157
100
156
100
154
100
153
100
153
100
155
90
159
90
158
90
157
90
157
90
158
90
156
80
151
Page 30 of 36
Raymond Delashmitt
2010 - 2011
60
60
60
60
60
50
50
50
50
50
2093
2086
2066
2040
1859
1648
1676
1733
1805
1732
Application of Fluorometers to Measure Wild Algal Growth In Vivo
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
1022
1021
1031
1035
1025
903
917
946
950
922
907
723
719
729
753
754
748
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
354
362
362
362
357
259
283
285
282
281
257
202
198
210
214
216
219
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
235
234
230
227
227
210
205
204
203
202
200
180
183
185
186
186
186
166
163
164
165
166
164
140
137
137
137
138
138
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
150
150
149
149
148
135
134
134
134
135
133
125
126
124
125
123
122
111
110
110
109
109
108
91
89
89
89
89
89
Page 31 of 36
Raymond Delashmitt
2010 - 2011
3 cm
Signal
Angle (Counts)
130
2317
130
2318
130
2302
130
2292
130
2286
130
2292
120
2383
120
2454
120
2458
120
2458
120
2467
120
2462
110
2469
110
2491
110
2492
110
2501
110
2500
110
2510
100
2498
100
2495
100
2499
100
2502
100
2502
100
2502
90
2488
90
2472
90
2464
90
2465
90
2464
90
2461
80
2432
80
2380
80
2377
80
2377
80
2377
80
2377
70
2410
Application of Fluorometers to Measure Wild Algal Growth In Vivo
In Beam Plane With Infinite Plane Source
4 cm
5 cm
6 cm
Signal
Signal
Signal
Angle (Counts)
Angle (Counts)
Angle (Counts)
140
1254
150
701
150
474
140
1249
150
779
150
480
140
1177
150
786
150
480
140
1215
150
780
150
478
140
1221
150
783
150
466
140
1229
150
780
150
481
130
1279
140
795
140
488
130
1290
140
796
140
492
130
1285
140
798
140
494
130
1309
140
807
140
500
130
1317
140
792
140
477
130
1319
140
794
140
469
120
1321
130
800
130
471
120
1338
130
814
130
469
120
1333
130
822
130
470
120
1322
130
818
130
472
120
1343
130
822
130
434
120
1327
130
807
130
438
110
1287
120
817
120
445
110
1286
120
813
120
446
110
1293
120
811
120
445
110
1291
120
816
120
448
110
1286
120
820
120
424
110
1268
120
793
120
419
100
1232
110
786
110
423
100
1235
110
771
110
426
100
1235
110
762
110
427
100
1230
110
765
110
428
100
1230
110
766
110
408
100
1199
110
731
110
396
90
1163
100
709
100
397
90
1164
100
711
100
397
90
1163
100
712
100
397
90
1162
100
713
100
397
90
1160
100
713
100
398
90
1122
100
679
100
372
80
1104
90
657
90
363
7 cm
Signal
Angle (Counts)
150
370
150
381
150
380
150
382
150
379
150
361
140
361
140
368
140
376
140
361
140
365
140
362
130
350
130
351
130
353
130
359
130
362
130
356
120
322
120
325
120
327
120
327
120
328
120
328
110
305
110
305
110
305
110
306
110
306
110
307
100
292
100
287
100
280
100
283
100
283
100
276
90
256
Page 32 of 36
Raymond Delashmitt
2010 - 2011
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
2428
2421
2418
2417
2417
2507
2542
2434
2429
2439
2446
2554
2615
2594
2589
2587
2583
Application of Fluorometers to Measure Wild Algal Growth In Vivo
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
1104
1103
1103
1102
1102
1106
1116
1095
1097
1097
1106
1115
1117
1115
1116
1084
957
785
751
688
754
780
669
577
610
644
642
642
90
90
90
90
90
80
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
30
30
30
30
30
639
633
639
639
639
607
596
596
594
595
599
577
577
577
578
580
578
570
568
560
560
561
562
554
530
532
522
521
524
499
491
478
477
478
482
421
377
370
367
361
90
90
90
90
90
80
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
30
30
30
30
30
365
364
364
363
348
343
344
345
346
345
346
340
338
338
339
340
344
345
352
355
356
355
353
335
331
316
318
322
361
376
365
319
332
338
326
251
229
225
225
224
90
90
90
90
90
80
80
80
80
80
80
70
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
40
40
40
40
40
40
30
30
30
30
30
255
254
255
255
250
227
228
230
231
230
229
214
213
213
212
212
210
194
187
186
184
184
183
160
163
162
162
162
160
125
129
132
132
133
133
101
102
102
103
104
Page 33 of 36
Raymond Delashmitt
2010 - 2011
3 cm
Signal
Angle (Counts)
120
2105
120
2104
120
2107
120
2106
120
2109
120
2111
110
2125
110
2134
110
2142
110
2147
110
2148
110
2152
100
2182
100
2189
100
2194
100
2195
100
2198
100
2201
90
2244
90
2254
90
2259
90
2260
90
2261
90
2260
80
2255
80
2260
80
2258
80
2261
80
2262
80
2262
70
2305
70
2332
70
2331
70
2329
70
2329
70
2329
60
2351
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Perpendicular to Beam Plane With Infinite Plane Source
4 cm
5 cm
6 cm
Signal
Angle (Counts)
130
1155
130
1153
130
1160
130
1155
130
1153
130
1153
120
1183
120
1189
120
1187
120
1189
120
1187
120
1187
110
1197
110
1198
110
1199
110
1194
110
1194
110
1200
100
1198
100
1200
100
1200
100
1199
100
1199
100
1200
90
1196
90
1196
90
1195
90
1194
90
1194
90
1193
80
1186
80
1185
80
1182
80
1179
80
1178
80
1175
70
1161
Signal
Angle (Counts)
130
627
130
624
130
595
130
602
130
609
130
611
120
617
120
618
120
618
120
617
120
618
120
618
110
617
110
616
110
617
110
618
110
618
110
617
100
613
100
610
100
612
100
612
100
613
100
612
90
607
90
607
90
607
90
607
90
607
90
607
80
613
80
611
80
608
80
608
80
608
80
609
70
611
Signal
Angle (Counts)
130
425
130
423
130
420
130
422
130
422
130
423
120
408
120
410
120
409
120
409
120
409
120
407
110
393
110
392
110
393
110
393
110
393
110
392
100
380
100
380
100
380
100
378
100
378
100
377
90
366
90
367
90
367
90
368
90
368
90
368
80
360
80
359
80
359
80
360
80
360
80
360
70
357
7 cm
Signal
Angle (Counts)
130
268
130
269
130
270
130
271
130
271
130
272
130
273
130
273
120
268
120
268
120
268
120
267
120
268
120
268
120
268
120
266
110
261
110
261
110
261
110
261
110
261
110
261
110
261
110
260
100
254
100
253
100
253
100
252
100
253
100
253
100
253
100
252
90
249
90
249
90
249
90
249
90
249
Page 34 of 36
Raymond Delashmitt
2010 - 2011
60
60
60
60
60
50
50
50
50
50
50
2355
2357
2356
2350
2356
2368
2350
2320
2320
2316
2299
Application of Fluorometers to Measure Wild Algal Growth In Vivo
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
1160
1160
1158
1156
1154
1123
1126
1123
1115
1109
1096
1004
999
996
994
996
984
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
607
604
604
603
604
596
596
592
593
594
593
587
591
590
589
590
589
70
70
70
70
70
60
60
60
60
60
60
50
50
50
50
50
50
357
357
356
355
355
343
343
343
344
344
344
342
341
339
338
338
336
90
90
90
80
80
80
80
80
80
80
80
70
70
70
70
70
70
70
70
60
60
60
60
60
60
60
60
50
50
50
50
50
50
50
50
249
249
248
244
243
244
244
243
243
243
243
238
238
238
238
238
238
238
236
227
227
228
228
227
227
227
225
218
219
219
219
219
220
219
219
Page 35 of 36
Raymond Delashmitt
2010 - 2011
Application of Fluorometers to Measure Wild Algal Growth In Vivo
Bibliography
Aberle, N. (September 2006). 'Spectral fingerprinting' for specific algal groups on sediments in situ: a
new senso. Stuttgart , 575-592.
H, U. (1958). Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Mitt. Internat. , 1-38.
SCCF Recon. (2010). Chlorophyll. Retrieved August 1, 2010, from SCCF Recon - Sanibel-Captiva
Conservation Foundation: River, Estuary and Coastal Observing Network:
http://recon.sccf.org/definitions/chlorophyll.shtml
Schroeder, W. &. (1994). Pigment patterns in suspended matter from Elbe and associated waters as
determined using high performance liquid chromatography. Neth. J Aquat. Ecol. , 255-265.
Wetlabs. (May 17, 2010). Characterization Sheet.
Wetlabs. (23 Dec 2009). Combination Fluorometer and Turbidity Sensor.
Page 36 of 36